A cytoplasmic based transcription system which uses a recombinant vaccinia virus expressing bacteriophage T7 RNA polymerase was described previously [Fuerst et al., Proc. Natl. Acad. Sci. USA 83, 8122-8126 (1986)]. When a plasmid containing a T7 promoter regulated gene was transfected into such vaccinia infected cells, the gene was transcribed and protein was made. Still higher expression was obtained when the T7 promoter regulated gene was incorporated into a second vaccina virus and cells were coinfected with the latter and the T7 RNA polymerase containing virus [Fuerst et al., Mol. Cell. Biol. 7, 2538-2544 (1987)]. A further improvement in expression was obtained by adding the encephalomyocarditis untranslated leader sequence downstream of the T7 promoter [Elroy-Stein et al., Proc. Natl. Acad. Sci. USA 86, 6126-6130 (1989)]. This permitted cap-independent translation.
In a previous study [Dunn et al., Gene 68, 259-266 (1988)], T7 RNA polymerase made in vitro was injected into monkey cells but the enzyme was not made in the cell nor was it shown to make functional mRNA. In another study, [Chen et al., Cell 50, 1047-1055 (1987)]yeast were transformed with the T7 RNA polymerase gene and RNA but no protein was made. In a very recent report, Deuschle et al. [Proc. Natl. Acad. Sci. USA 86, 5400-5404 (1989)]made a stable transformed rabbit cell line that expressed T3 RNA polymerase which is very similar to T7 RNA polymerase. DNA transfected into these cells was transcribed but no protein was detected unless the cells were infected with vaccinia virus which presumably supplied capping enzyme needed for translation.
The present invention represents a major improvement in the field and involves the construction of a stable mammalian cell line that expresses a foreign RNA polymerase gene. For transient expression assays, this cell line eliminates the requirement for the vaccinia virus containing the T7 RNA polymerase gene. This is important because vaccinia virus causes a cytopathic effect and is also a health hazard. Alternatively, the cell line can be infected with a single recombinant vaccinia virus containing the T7 promoter regulated foreign gene and dispense with the requirement for a second vaccinia virus containing the T7 RNA polymerase. This simplifies the expression system particularly for large scale work in which it is inefficient to infect cells with two viruses simultaneously.